Identification of non-procyclin molecules expressed
by
Trypanosoma
brucei brucei
procyclic culture forms
Emily Jansen
B. Sc., University of Victoria, 200 1
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of
MASTERS OF SCIENCE
in the Department of Biochemistry and Microbiology
O Emily Jansen, 2005 University of Victoria
A11 rights reserved. This thesis may not be reproduced in whole or in part, by photocopy or other means, without the permission of the author.
Supervisor: Dr. Terry W. Pearson
Abstract
African trypanosomes cause sleeping sickness in humans and nagana in cattle, both economically devastating diseases in Sub-Saharan Africa, for which a vaccine remains elusive. Trypanosomes exhibit a complex, digenetic life cycle and alternates between the tsetse fly vector (procyclic form) and a mammalian host (bloodstream form). It has proven dificult to study less abundant surface molecules expressed by African trypanosomes because procyclins andlor variant surface glycoproteins (VSG) form a physical barrier that prevents antibodies and labeling reagents from binding to invariant, membrane-embedded molecules. Creating a VSG knock out in bloodstream form trypanosomes has not been accomplished. However, procyclin knock out mutants have been generated that do not express the major surface-disposed procyclins. Other mutants have been generated that do not express any GPI-anchored proteins on their surface. Analysis of protein profiles by 1D gel and 2D gel electrophoresis of wild type, KO1 JEPIGPEET knock out), Now 6 (EP knock out) and KO2 (GPIIO knock out) pxocyclic culture forms of T. b. brucei revealed no differences between the proteins expressed by these parasites. Monoclonal antibodies (mAbs) were generated against the two procyclin knock out mutants, KO1 and Nour 6. After preliminary screening of hybridoma supernatants for mAbs that recognized surface antigens, mAbs 1G10 and 2E1 (anti- KOl), and mAbs 1C3 and 2G4 (anti-Nour 6) were selected for further analysis using immunofluorescence microscopy, flow cytometry and immunoblotting. Using 2D gel electrophoresis and immunoblot analysis in combination with MALDI-TOF mass spectrometry, the antigens recognized by mAb 1G10 were identified as a- and P-tubulin fiom T. b. rhodesiense. MAbs 2E1, 1C3 and 2G4 did not show irnrnunoblot activity. Various attempts to identifl the antigens were unsuccessful. However, experiments using the KO2 mutant which, does not express any GPI-anchored surface molecules, allowed tentative identification of some of the antigens as potential GPI-anchored surface molecules. Surface biotinylation with Sulfo-NHS-biotin of wild type, K01, Nour 6 and KO2 procyclic culture forms of T. 6. brucei indicated that in addition to the procyclins
these parasites express different surface protein profiles. Biotinylation, in combination with avidin affinity chromatography, was used to isolate surface antigens expressed by KO1 knock out PCF trypanosomes. Two dimensional gel electrophoresis and MALDI- TOF mass spectrometry were used in attempts to identify these surface molecules. The results identified a- and f3-tubulin from T. b. rhodesiense, in addition to paraflagellar rod proteins, a putative coatomer f3-subunit, a probable adenylatelguanylate cyclase and an unknown hypothetical protein flom T. b. brucei.
Table of contents
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... iv , . .List of Tables
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ix . .List of Abbreviations
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General introduction
... 1I. African trypanosomiasis; the disease ... 1
i; A brief history of African sleeping sickness ... 2
ii. Disease prevalence and its impact today ....
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.611.' Diagnosis and Chemotherapeutic treatment for African sleeping sickness .
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.7i,. Diagnostic tests for African sleeping sickness . ... ... ...
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.... .... ... .. .. .. ... .7ii. Chemotherapeutics for Afkican sleeping sickness ... ... 8
iii. Drug resistance and future alternatives for control of African trypanosomiasis. ... 12
III. Trypanosome biology ... ...
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.16ii. Parasite life cycle in the tsetse fly vector ... 19
iii. Parasite life cycle in the mammalian host ... ... ....
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The surface coat of PCF African trypanosomes
... 271
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Introduction ... 271.1 Variant surface glycoproteins of bloodstream forms of African ... trypanosomes 27 i
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Regulation of VSG gene transcription ... 27ii
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Structure of VSG proteins ... 28... iii
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Functions of VSG 30 1.2 Procyclins expressed by PCF African trypanosomes ... 30i
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The discovery of procyclins ... 31... ii
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The structure ofprocyclins 32 ... iii.
Functions ofprocyclins 33 iv.
Regulation of procyclin expression ... 351.3 Glycoconjugates of other procyclic stage trypanosomes ... 36
1.4 Non-variant surface molecules ... 37
i
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The transferrin receptor ... 38ii
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Adenylate cyclase receptor ... 39... ul
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Lipid receptors ... 40 ... iv.
Glucose transporters 41 ... v.
Membrane boundproteases 42 ....
vi Ion pumps 44 vii.
Purine transporters ... 45...
1.5 Critical roles of GPI-anchors for
T.
bmcei 46...
1.6 Hypothesis -50
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1.7 Research strategy 50
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2
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Materials and Methods 53...
2.1 Trypanosomes 53
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2.2 Cryopresewation of Trypanosomes 54
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2.3 One dimensional polyacrylamide gel electrophoresis 54
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2.4 Two dimensional polyacrylamide gel electrophoresis 55
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2.5 Colloidal Coomassie Brilliant Blue 6-250 staining of proteins in gels 55
2.6 Silver nitrate staining of proteins in gels ... 56
2.7 Membrane preparation ... 57
2.8 BCA protein assay of membrane fractions ... 58
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... 2.9 Immuniza~ons 58 2.10 Enzyme-linked immunosorbent assay (ELISA) ... 591.11 Derivation of monoclonal antibodies ... 60
2.12 Cryopreservation of hybridomas ... 61
2.13 Isotyping of monoclonal antibodies
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62... 2.14 PEG purification of ascites mAb 1610 62 ... 2.15 Immunofluorescence microscopy and flow cytometry 63 2.16 1-D and 2-D immunoblot analysis ... 64
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2.18 Mass Spectrometry -66
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2.19 Biotin labeling of cell surface proteins ... 67 2.20 Avidin affinity chromatography
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3
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Results 69... 3.1 1D and 2D protein profiles of wild type and mutant
T.
b.
brucei PCF 69...
3.2 Membrane preparation from EP/GPEET KO1 trypanosomes 73...
3.3 Derivation of monoclonal antibodies 73
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3.4 Selection of monoclonal antibodies 76
3.5 Immunofluorescenct microscopy and flow cytometric analysis of mAbs
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lGlO and 2E1 76
... 3.6 1D and 2D immunoblot analysis of mAbs 1GlO and 2E1 82
... 3.7 MALDI-TOF analysis of antigens recognized by mAb 1610 83 3.8 Specificity of mAb 1GlO ... 90
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3.9 Analysis of mAbs 1C3 and 264 92
3.10 Analysis of biotin labeled proteins of
T.
b.
brucei PCF trypanosomes .. 99...
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4 Discussion.. 108
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Bibliography...
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Appendix I ... 133. . . V l l l
List of
Tables
Table 1. Summary of proteins identified by MALDI-TOF mass spectrometry analysis of protein spots recognized by mAb lGlO ... .
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.89Table 2. Summary of proteins identified by MALDI-TOF mass spectrometry analysis of biotinylated proteins separated by 2D gel electrophoresis . . . .I06
List of Figures
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.
Figure 11.
Figure 12.
Figure 13. Figure 14.Geographical distribution of African sleeping sickness ... 5
Principal cellular structures of T. b
.
brucei ... 16Digenetic life cycle of African trypanosomes ... -20
Major surface molecules of BSF and PCF T. b
.
brucei ... 29Structures of EP and GPEET procyclins ... 34
GPI biosynthesis of procyclins in
T.
b.
brucei ... 47Schematic representation of wild type and three surface knock out mutants of T. b
.
brucei PCF ... 51One-dimensional gel electrophoretic separation of proteins of wild type and mutant PCF T. b
.
brucei stained with Colloidal Coomassie Blue ... 70One dimensional gel electrophoretic separation of proteins of wild type and mutant PCF
T.
b.
brucei stained with silver nitrate ... 71Two-dimensional polyacrylamide gel electrophoresis of proteins from ... wild type and mutant T. b
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brucei PCF 72 One-dimensional gel electrophoretic separation of membrane proteins extracted from T. b.
brucei KO1 knock out PCF...
74Protein quantification of membrane preparations of
T.
b.
brucei PCF ... 75Indirect ELISA titration of test bleed serum from immunized mice ... 77
Flow cytometric analysis of fixed wild type and KO1 PCF labeled with anti-KO 1 rnAbs 1 G 10 and 2E1 ... -79
Figure 15. Flow cytometric analysis of live wild type and KO1 PCF labeled with anti-KO 1 mAbs 1 G 10 and 2E 1 ... .80
Figure 16. Immunofluorescence analysis of fixed wild type and mutant procyclic ... trypanosomes labeled with mAb 1 G 10 and 2E 1.. -8 1
Figure 17. Immunoblot analysis of wild type and KO1 T. b. brucei PCF with mAb lGlO and 2E1 ... 84 Figure 18. Detection of tubulin in wild type T. b. brucei PCF afier 2D gel
electrophoresis and immunoblotting
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-85Figure 19. 2D gel profile showing protein spots extracted for MALDI-TOF analysis to identifjr the antigen recognized by mAb 1 G 10 ... -86
Figure 20, Peptide mass spectrum of the 60 kDa protein recognized by mAb 1 GI0 . .88
Figure 21. Detection of tubulin in wild type and knock out PCF mutant trypanosomes ... -9 1
Figure 22. Flow cytometric analysis of live wild type and KO1 PCF labeled with mAbs 1C3 and 2G4 ... .93
Figure 23. Immunofluorescence analysis of live wild type and mutant procyclic
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trypanosomes labeled with mAbs 1 G10 and 2E 1 .94
Figure 24. Immunofluorescence analysis of fixed wild type and mutant procyclic
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trypanosomes labeled with mAbs 1 GlO and 2E1 .95
Figure 25. Immunoblot analysis of wild type and KO1 T. b. brucei PCF with mAbs
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1C3 and 2G4 98
Figure 27. Biotinylated proteins of KO1 knock out PCF trypanosomes isolated by streptavidin chromatography and separated by 2D SDS-PAGE ... .lo5
xii
List of Abbreviations
AEBSF ATP BCA BSF ID 2D 3D CAMP CHAPS DFMO DMSO DTT ELISA EP ER ES ESAG FBS FP GlcNAc GPEET GPI GPIl 0 GP18 GPI-PLC GPI-PLD HCl HDL HEPESm o
IgM ISG kDa KO1 KO2 LDL rnAb MALDI-TOF MSP NGO Nour 6 NHS 4-(2-aminoethy1)benzenesulfonyl fluoride adenosine triphosphate bicinchoninic acid bloodstream form one dimensional two dimensional three dimensionalcyclic adenosine monophosphate
(3
-
[(3 -cholamidopropyl)dimethylammonio]- 1 -propanesulfonate) difluoromethy lornithinedimethyl sulfoxide dithiolthreitol
enzyme linked immunosorbent assay glutamate-proline
endoplasmic reticulum expression site
expression site associated gene fetal bovine serum
flagellar pocket N-acety lglucosamine
glycine-proline-glutamate-glutamate-threne glycosylphosphatidylinositol
third mannosyl transferase gene protein transamidase gene GPI-specific phospholipase C GPI-specific phospholipase D hydrochloric acid
high density lipoprotein
(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfoonic acid] horseradish peroxidase
immunoglobulin isotype G immunoglobulin isotype M invariant surface glycoprotein kiloDalton
EP/GPEET procyclin knock out T. b. brucei GPI 10 knock out
T.
b. bruceilow density lipoprotein monoclonal antibody
matrix assisted lazer desorption ionization-time of flight major swrface protease
non-governmental organization EP procyclin knock out T. b. brucei
..* X l l l OD PBS PCF PEG PIC PMSF
PW
PVDF RNAi SDS-PAGE TIFF THT TLF Tf-R VSG WHO optical densityphophate buffered saline procyclic culture form polyethylene glycol protease inhibitor cocktail phenylmethanesulfonyl fluoride p-nitrophenyl phosphate
polyvinylidene fluoride ribonucleic acid interferance
sodium dodecyl sulfate-polyacrylamide gel electrophoresis tag image file format
trypanosome hexose transporter trypanosome lytic factor
transfenin receptor
variant surface glycoprotein world health organization
xiv
Acknowledgements
I would like to take this opportunity to thank the people who have contributed to this research by sharing their knowledge and expertise and to the people who have given me their love and support. This experience has enriched my life and left its mark. I will always have a special place in my heart for parasites and Afiica. I would like to first off thank Terry Pearson for his inspiring lectures in fourth year immunology that lead me to travel in Africa and upon return request the opportunity to join his lab as a graduate student. Thankfully he said "yes" and I greatly appreciate the opportunity he gave me to study trypanosomes in his lab. I would like to thank Jody Haddow and Lee Haines for teaching me the ropes when I first came to the lab, and for sharing their multitude of knowledge and skills with me over the past three years. I would like to thank Angela Jackson for being such an enthusiastic and friendly co-student with whom it has been a pleasure to share the Grad school experience with. I thank Darryl Hardy and Derek Smith for their expertise in proteomics, a tool that greatly contributed to this research. I thank Stephen Horak and Scott Scholtz for there technical expertise with lab equipment and computers that greatly facilitated my research, and peace of mind, throughout my degree. I would like to thank Michael Grigg for h s scientific consult over the past two years and for h s contagious enthusiasm towards parasites. I would like to thank Deanna Gibson for being my ear through all these years at U.Vic, her companionship and constant support have kept me sane! I also thank Christina Sales for her invaluable friendship and support. I would like to thank Tan Lee for her understanding, love and support while writing this thesis, it was very much appreciated. Last but definitely not least I thank my parents, Karen Moon and Frans Jansen, and my sister Jessica Jansen, for their unconditional love and support that I always knew I could count on.
Chapter 1. General introduction
I. African trypanosomiasis: the disease
Two subspecies of African trypanosomes cause two different clinical forms of sleeping sickness in humans (Barrett et al., 2000). In West Africa, Trypanosoma brucei gambiense is the causative agent of a chronic form of the disease. In contrast, in East Africa, Trypanosoma brucei rhodesiense causes an acute form of the disease (Barrett et al., 2000). African sleeping sickness consists of a two stage pathological evolution and, if left untreated, both the acute and chronic forms of the disease are fatal (Buguet et al., 2001). Trypanosomes induce an inflammatory reaction at the site of infection and a skin chancre develops that resolves within one month (Vickerman, 1993). This is the haemolymphatic stage I phase in which the parasites quickly disseminate to the lymphatic system and bloodstream, spreading throughout the body (Vickerman, 1985; Buguet et al., 2001). This early stage is characterized by general malaise, waves of fever, joint pain, peripheral oedema, anemia, headaches and swollen lymph nodes in the neck (Barrett et al., 2000; Burchmore et al., 2002). This can be accompanied by myocarditis, pulmonary oedema, pericardial efhsion, ascites, splenomegaly and hepatomegaly (Burchmore et al., 2002). Continuous waves of parasitemia provoke inflammatory reactions within various tissues and organs that have been invaded by the parasites. Stage I ends when the parasites invade the spinal cord and brain. Progression to the neurological stage I1 phase usually occurs within the first month of a T. b. rhodesiense infection but may take several months or years to develop in a T. b. gambiense infection (Barrett et al., 2000; Buguet et al., 2001; Burchmore et al., 2002). Stage I1 can be diagnosed by the presence of parasites
andlor mononuclear inflammatory cells in the cerebrospinal fluid (Barrett et al., 2000; Buguet et al., 200 1). Parasite invasion of the central nervous system (CNS) occurs via an unknown mechanism and is accompanied by lymphocyte infiltration, associated vasculitis and perivascular cuffing (Buguet et al., 2001; Burchmore et al., 2002). The disease now manifests itself by causing severe headaches, personality changes, sleep disorders and weight loss, eventually leading to brain function deterioration, seizures, coma and ultimately, death (Black et al., 2001; Burchmore et al., 2002; Stich et al., 2003).
In addition to their great impact on human health, African trypanosomes also cause disease in livestock including cattle and other ruminants, pigs, horses and dogs. The pathogens of animal trypanosomoses are Trypanosoma brucei brucei, Trypanosoma vivax and Trypanosoma congolense which cause disease in cattle (nagana, from the Zulu meaning "poorly"), Trypanosoma simiae which is responsible for high mortality in pigs. In addition to causing disease in cattle Trypanosoma brucei brucei affects all livestock, with a particularly high mortality incidence in horses and dogs. Nagana has had a huge impact on agricultural development and socio-economic growth in endemic countries. This is mainly due to the critical role of livestock, primarily cattle, in farming throughout Sub-Saharan Africa (Aksoy et al., 2003).
i. A brief history of African sleeping sickness
Hundreds of years passed between the recognition by slave-traders in West Africa that slaves with swollen lymph nodes at the back of their necks would likely die on the crossing to America, and identification of the parasites that caused this disease. At the
beginning of the twentieth century, the identification of trypanosomes as the causative agent of sleeping sickness was surrounded by controversy. In 1903 Aldo Castellani, working in Uganda, observed a parasite in the cerebral spinal fluid of a patient. However, he did not initially claim this parasite to be the cause of sleeping sickness. David Bruce thought differently and supposedly (as he claimed after the publication) he was the one who convinced Castellani that the parasite might indeed be the culprit. Castellani was given official credit as the sole publisher of this discovery and in the same year Bruce identified the tsetse fly as the active vector (Stebeck et al., 1994)
.
Trypanosomes were to play an integral role in the shaping of Africa and its colonization. A description of sleeping sickness dates as far back as the fourteenth century when Ibn Khaldoun in his "History of the Berbers" wrote that the Sultan Djata of the Kingdom of Melli (now Mali) was stricken by a wasting lethargy disease that killed him. Caravanners recognized signs of sleeping sickness in travelers from the southern kingdoms long before they knew the origin or cause. It was not until the twentieth century that particularly severe epidemics occurred in Africa, killing millions and stimulating thorough and extensive control campaigns in afflicted areas. At the turn of the century, from 1896 to 1906, a massive epidemic raged in Uganda and the Congo basin, leaving half a million dead. The second major epidemic was during the 1920's and the third began in the 1990's and continues today (WHO, 2005).
During the second major epidemic the disease was rampant throughout east, south and West Africa. By this time the relationship between trypanosomes, the tsetse fly vector and sleeping sickness had been discovered and drugs were developed to treat the disease. The Sleeping Sickness Bureau, involving multiple countries, pulled together to
initiate a campaign to bring the disease under control. This included treatment of patients and vigilant, continuous screening to decrease the size of the human reservoir that was responsible in part for the rapid transmission. In addition, methodical clearing of undergrowth from villages, water sources, paths and bridges was performed to decrease the natural habitat of the tsetse fly. With the continuous screening of millions of people each year in endemic areas, the number of new cases per year fell and by 1960 the disease was basically eliminated. It took nearly half a century to bring down the disease. Active screening was then largely discontinued and within the last two decades Africa has seen a reemergence of the disease (htt~://www.who.int/ernc/diseases/tr~~).
ii. Disease prevalence and its impact today
African independence began within an era of greatly reduced or complete absence of sleeping sickness. However, many of the newly formed countries did not have the human or financial resources to keep up the critical monitoring required to keep the disease in check. Along with the discontinued use and breakdown of disease control programs, and with increased civil unrest, war, famine and the emergence of HIV, it is not surprising that a new epidemic of sleeping sickness began in the 1970's, reached severe epidemic proportions in the 1990s and continues today. African sleeping sickness is now considered a major health problem in sub-Saharan Africa.
African sleeping sickness threatens 60 million people within 36 countries in sub- Saharan Africa (Figure 1) (Aksoy et al., 2003). Only three to four million people in endemic areas are under active surveillance and therefore the numbers reported do not reflect the reality of the situation. It is estimated that the number of people currently
afflicted with the disease is between 300,000 and 500,000. In Angola, Central African Republic, Sudan and the Democratic Republic of Congo current epidemics rage with a prevalence of 20-50% making sleeping sickness the leading cause of mortalit
HW/AIDS) in these areas (Figure 1) (http://www.who.int/emc/diseases/tryp).
Trypornosomiasis NoRisk
0
At Riska
EndemicHigh ~ n d e m i c
Figure 1. Geographical distribution of Afiican sleeping sickness. T. b. gambiense causes chronic disease
in
West and Central Afi-ica and T. b. rhodesiense causes acute disease in East Africa. This is illustrated by the black line that separates sub-Saharan Afi-ica into the two different parasite zones. The legend depicts a scale that goes from areas that have no risk of disease toareas experiencing an epidemic (WHO 2004).
(httQ://www.rnedicalecolow .or~/diseases/d afiican trypano. htm)
(ahead of
the Afkican trypanosomes that cause nagana in livestock, T. vivax and T. congolense are the most important and are responsible for massive economic losses,
-
US$ 1338 million annually @avila, 2003). There are 200 million cattle at risk within sub-Saharan Africa (Aksoy et al., 2003). The trypanosomes are not only criticaleconomically but are socially devastating as well, causing a decrease in the labor force and abandonment of fertile land where the disease is rife (Stebeck et al., 1994).
iii. Geographical distribution and Transmission
Except for T. vivax, which can be mechanically spread by biting insects, and T. equiperdum, which is sexually transmitted in horses, transmission of most trypanosomes is strictly dependent upon the tsetse fly insect vector. Consequently, the trypanosomes are restricted to sub-Saharan Africa between the latitudes of 14 ON and 29 "S where the tsetse fly population thrives (Figure 1) (Ruepp et al., 1997).
Transmission occurs mainly through the bite of an infected tsetse fly, although it is also possible to contract the disease from contaminated blood, shared needles and in- utero acquisition through the placenta (htt~://www.who.int/emc/diseases/trv~). There are 3 1 members of the Glossina family and both male and female tsetse flies can transmit the disease (Aksoy et al., 2003). There are seven species of tsetse fly that transmit human sleeping sickness and they are all unique to sub-Saharan Africa. Three Glossina species, all of the palpalis group, act as vectors for T. b. gambiense: G. palpalis, G. fuscipes and G. tachinoides. Four Glossina species of the morsitans group are vectors for T. b. rhodesiense: Glossina morsitans morsitans, G. morsitans centralis, G. swynnertoni and G. pallidipes. Tsetse flies require warm, shady, and humid areas to survive and reproduce, therefore restricting the disease to areas in sub-Saharan Africa where these particular conditions can be found. A tsetse fly usually lives for six months and once infected by trypanosomes, remains a vector for life. Tsetse flies that carry T. b. rhodesiense live primarily in the savannah woodlands of eastern and southern Africa and
come in contact with humans going about their daily activities such as gathering wood, hunting, fishing, farming and herding cattle. T. b. gambiense causes sleeping sickness mainly in lowland rain forests of West and Central Africa and is spread primarily by peri- domestic tsetse flies living in areas surrounding human habitats, such as cultivated land and near small rivers or pools of water, frequented by people. Therefore it is easy to see why this disease is so readily and widely spread (http://www.who.int/emc/diseases/trvp).
11. Diagnosis and Chemotherapeutic treatment of African sleeping sickness
i. Diagnostic tests for African sleeping sickness
Traditional diagnosis was performed using microscopic analysis to visualize trypanosomes in the blood and other body fluids such as cerebrospinal fluid. However, this is unreliable due to the fluctuating numbers of parasites seen throughout the infection (WHO, 1979). Anti-trypanosome antibodies found in the serum are often more reliable, although an active infection, and a cured infection, cannot be distinguished (WHO, 198 1). The indirect immunofluorescence antibody test has been the most commonly used diagnostic tool in human sleeping sickness surveys. For this test, blood is collected and eluted fkom filter paper and used for detection of invariant antigens in acetone-fixed trypanosome smears. Another widely used test for the Gambian form of sleeping sickness is the card agglutination trypanosomiasis test. For this test, a drop of fresh blood is mixed on a card with a drop of fixed, stained trypanosome suspension. A positive test will show macroscopic agglutination within two minutes. Monoclonal antibodies have been developed that distinguish between cattle infections with T. brucei sspp, and with T.
congolense and T. vivax (Vickerman, 1 993). More recently, an indirect enzyme-linked immunosorbent assay (ELISA) was used to detect titers of anti-T. brucei antibodies in blood collected fiom primates (Jeneby, 2002). A new form of ELISAs called PCR- ELISAs using species-specific primers has been used to identify T. vivax or T. brucei infections in cattle (Masake, 2002). Diagnostics are much needed to distinguish between the different stages of disease, as the stage determines the type of treatment. Recently, a test has been devised to identify the stage I1 forms of disease using IgM intrathecal detection to identify CNS involvement. A latex agglutination test has also been developed which is more practical to use in the field (Lejon, 2002).
ii. Chemotherapeutics for African sleeping sickness
There are no vaccines for African trypanosomiasis; therefore drugs remain the principle means of intervention. Over the past century, there have only been four drugs developed to treat African sleeping sickness, although in the past few years several new ones have been identified and are now in clinical trials. With the standard drugs, there are several problems including: toxicity, increasing drug resistance, administration difficulties, and expense. The drug of choice depends on the infective sub-species of T. brucei and whether or not the disease is diagnosed prior to invasion of the central nervous system. The four drugs are: 1) Suramin, 2) Pentamidine, 3) Melarsoprol and 4) Difluoromethylornithine (DMFO).
1) Suramin was first introduced in 1922 and is a polyanionic sulfonated naphylamine, chemically related to Paul Ehrlich's trypan red and other similar naphylamine dyes with trypanocidal activity (Barrett et al., 2000; Denise et al., 2001 ;
Burchmore et al., 2002; Fairlamb, 2003). Today it remains the treatment of choice for early stage T. b. rhodesiense infections, despite its multitude of side effects. These include: nausea, vomiting, rash, fever, shock, collapse and renal damage (De Koning, 2001; Burchmore et al., 2002; Fairlamb, 2003). No significant resistance to the drug has emerged within the last 80 years. However, it has a 25-35% treatment failure rate (Burchmore et al., 2002; Fairlamb, 2003). Its mode of action remains obscure even after many years of investigation. Suramin contains six negative charges at physiological pH and therefore it has a high avidity for serum proteins including low density lipoprotein (LDL) (Barrett et al., 2000; De Koning, 2001 ; Denise et al., 2001 ; Burchmore et al., 2002; Fairlamb, 2003). Trypanosomes have a receptor for low density lipoprotein (LDL) and it has been hypothesized that LDL is used to accumulate the drug via receptor- mediated endocytosis, although this has recently been disputed (Denise et al., 2001; Burchmore et al., 2002; Fairlamb, 2003).
2) Pentamidine was discovered after a similar compound, (synthalin), which induces hypoglycaemia in mammals, was found to have profound anti-trypanosomal activity (Denise et al., 2001; Burchmore et al., 2002; Fairlamb, 2003). This drug has been in use for over 50 years and was introduced in the 1940's as a trypanocidal chemotherapeutic (Burchmore et al., 2002; Fairlamb, 2003). Treatment is restricted to the early stage of T. b. gambiense infections because the drug does not readily cross the blood-brain barrier to the central nervous system. It is not understood why it fails with T.
b rhodesiense infections (Barrett et al., 2000; Denise et al., 200 1 ; Burchmore et al., 2002; Fairlamb, 2003). Side effects include: nausea, severe hypotensive reactions following injection, liver and kidney damage (nephrotoxicity) and pancreatic damage, which can
lead to diabetes (Burchmore et al., 2002; Fairlamb, 2003). This drug has poor oral bioavailability and must be administered by peritoneal injection making it difficult to administer in the field. Despite broad use of this drug in attempts to eradicate disease caused by T. b. gambiense during the 1950's and 1960's, widespread resistance has not emerged in the field (Fairlamb, 2003). The multiple means by which pentamidine is taken up by the parasite may account for the lack of drug resistance (Barrett, 1999; De Koning, 200 1; De Koning, 2001; Denise et al., 2001; Fairlamb, 2003). Pentamidine is an aromatic diamidine that acts directly against the parasites, independent of their physiological action on the host (Barrett, 1999; Barrett et al., 2000; De Koning, 2001; De Koning, 2001; Denise et al., 2001; Burchmore et al., 2002; Fairlamb, 2003). The mechanism of action is not well understood, although it is known to be taken up by at least three transporters (P2, HAPTl and LAPT1) and accumulated to millimolar concentrations within the cells (Barrett, 1999; Hanna, 2000; De Koning, 2001; De Koning, 2001; Denise et al., 2001; Burchmore et al., 2002; Fairlamb, 2003).
3) Melarsoprol was developed based on information gained from studies of arsenicals, first introduced by Paul Ehrlich at the turn of the century (Barrett et al., 2000; Denise et al., 2001; Burchmore et al., 2002). Melarsoprol was introduced to the market as an anti-trypanosomal drug in 1949. It is the only drug available to treat late stage T. b. gambiense and T. b. rhodesiense infections and resistance has been reported in the field (Barrett et al., 2000; Denise et al., 2001; Burchmore et al., 2002; Fairlamb, 2003). Melarsoprol is water insoluble and must be given intravenously dissolved in propylene glycol, a highly irritable solvent (Fairlamb, 2003). Of all the trypanocidal drugs, melarsoprol causes the most severe toxic effects including encephalopathy in 5-10% of
cases, of which half are fatal (Burchmore et al., 2002; Fairlamb, 2003). Other common side effects include: vomiting, abdominal colic, peripheral neuropathy (seizures), arthralgia, and thrombophlebitis (leakage of propylene glycol from the site of injection into surrounding tissue) (Burchmore et al., 2002; Fairlamb, 2003). The mechanism of the severe and potentially fatal encephalopathy is unknown but does not seem to be restricted to patients treated for sleeping sickness (Fairlamb, 2003). Trivalent arsenicals are promiscuous inhibitors of many enzymes or substrates that contain vicinal thiol groups. Trypanosomes express a specific purine transporter and loss of this transporter leaves trypanosomes resistant to Melarsoprol. Once within the cell, the arsenical drug could potentially inhibit many vital metabolic and transport functions, which could lead to loss of motility and lysis which parasites experience when treated with these drugs (Burchmore et al., 2002; Fairlamb, 2003).
4) DMFO was first developed as an anti-cancer reagent. However, even though it remains at the trial stage for treatment of neoplastic disease, it does exhibit activity against both early and late stage T. b. gambiense sleeping sickness (Barrett et al., 2000;
Denise et al., 2001; Burchmore et al., 2002; Fairlamb, 2003). Most cases do not respond effectively and therefore DFMO is not used for treatment of T. b. rhodesiense sleeping
sickness (Denise et al., 2001; Fairlamb, 2003). This drug is far from ideal as it is very costly ($750/patient) and difficult to administer because it requires four daily infbsions of 400 mg/kg/day over a 2 hour period for 7-14 days (Barrett et al., 2000; Burchmore et al., 2002; Fairlamb, 2003). Side effects of DFMO are minimal, however diarrhea, anemia and other blood cell reductions are known. The lack of toxic side effects is due to the mechanism by which this compound elicits its trypanocidal activity. DFMO is an analog
of ornithine and acts as a specific, irreversible inhibitor of the enzyme ornithine decarboxylase (ODC), the first committed step in the biosynthetic pathway of polyamines. DFMO has similar specificity for both parasite and mammalian ODC. However, mammalian cells are able to replenish ODC much faster than trypanosomes, therefore a pulse of DFMO can deprive trypanosomes of ODC for a long period of time compared to mammalian cells, leading to cessation of parasite growth (Barrett et al., 2000; Denise et al., 2001 ; Burchmore et al., 2002; Fairlamb, 2003).
iii. Drug resistance and future alternatives for control of African trypanosomiasis Treatment of sleeping sickness is limited to a very small number of drugs that are expensive, difficult to administer in the field, and toxic. In addition, they are becoming increasingly ineffective due to the emergence of drug resistant parasites. With the resurgence of trypanosomiasis across Africa and the increase in arsenic resistant parasites, the need for new anti-trypanosomal drugs has never been greater. There are huge obstacles to overcome before new drugs are developed and put into use. These include the stringency of licensing criteria that will not allow potential drugs such as megazol to proceed to final trials because it does not pass the Arnes test, even though it has cured Simians of trypanosomiasis with a single dose and exhibited no side effects (Denise et al., 2001). Pharmaceutical companies are reluctant to invest in development of new drugs for sleeping sickness because of the unlikely return of their investment from markets in developing countries. This often leaves academia responsible for drug development (Denise et al., 2001; Davila, 2003). Human African sleeping sickness seems to have received a great deal of scientific interest when compared to other
diseases. However, this is misleading because African trypanosomes have become a paradigm for research into basic biology and eukaryotic cell function due to several unique characteristics with respect to their biochemical composition and gene regulation (Stich et al., 2003). Therefore, the bulk of research focused on trypanosomes does not encompass studies on diagnosis, treatment or control (Stich et al., 2003). This paints a bleak picture. However, three initiatives have been put into motion aimed at bringing the disease back under control (Stich et al., 2003). First, the International Atomic Energy Agency (IAEA) announced their plan to implement a continent-wide release of sterile tsetse. This followed a successful trial on Zanzibar where tsetse trapping, insecticide spraying and release of sterile males led to the complete eradication of tsetse from the island (Stich et al., 2003). This goal seems unrealistic, as it cost millions of dollars just to eradicate tsetse from Zanzibar, a small isolated island, whereas the tsetse belt in sub- Saharan Africa covers an area greater than the United States of America. Second, the World Health Organization (WHO) and Medecins Sans Frontieres (Doctors Without Borders) initiated a campaign to encourage two pharmaceutical companies to re-engage in production of drugs for sleeping sickness. This was done after the "DFMO scandal" which occurred when DFMO was no longer produced in the formulation needed for treatment of human sleeping sickness but instead appeared as a facial depilatory cream. After much outcry, Aventis agreed to donate pentamidine, melarsoprol and DFMO in 2001. Bayer then committed to continued production of suramin (Stich et al., 2003). Third, a major funding initiative by the Bill and Melinda Gates Foundation has enabled one compound, the pro-drug DB 289, a dicationic compound, to enter clinical trials (Stich
et al., 2003). The DB 289 compound has proven to be extremely effective and is now entering Phase I11 clinical trials (http://www.immtech.biz~african.html).
In countries most affected by the resurgence of sleeping sickness (Sudan, Central African Republic, Democratic Republic of Congo and Angola) control options exceed the capacities of the responsible governmental institutions. This is due to a combination of lack of funds, movement of people, famine, drought, civil unrest andlor war which lead to a breakdown of even the most basic infrastructure (Abel et al., 2004). In many places, such as Angola, NGOs have intervened, in an attempt to help bring the disease back under control (Abel et al., 2004). African sleeping sickness is a public health problem in countries where research infrastructure barely exists, hence, the absence of scientific research to answer hndamental questions in the understanding of disease pathogenesis, clinical presentation, effective control approaches, diagnostics and treatments (Abel et al., 2004). The disease, which is often present at very low levels, returns as soon as active surveillance is abandoned, usually due to war, lack of security, and poverty.
It is obvious from the resurgence of sleeping sickness that new control methods are required along with more consistent surveillance of endemic areas. One approach is the screening of new trypanocidal compounds to develop new drugs for treatment. Ideally these would be inexpensive to manufacture, less toxic than current drugs, orally administered and designed to be refractory to biochemical mechanisms responsible for drug resistance. An alternative approach to drugs that target the parasite, is the use of sterile insect technology ie. approaches involving tsetse eradication by release of sterile males. Another approach that is gaining popularity is the genetic engineering of symbiotic bacteria to manipulate vector-parasite interactions. It has been shown that
production of parasite-inhibitory molecules by vector symbionts greatly reduces parasite development and viability within the tsetse fly. This approach is called paratransgenesis. Tsetse flies naturally harbor bacterial symbionts that could theoretically be altered to express a foreign gene product. The primary symbiont Wigglesworthia glossinidia lives in specialized cells called bacteriocytes found in the bacteriome whereas the secondary symbiont, Sodalis glossinidius lives both freely in the hemolymph and midgut and intracellularly in midgut epithelial cells. The midgut is an ideal compartment to inhibit trypanosome development, differentiation or transmission. In the midgut bloodstream form (BSF) trypanosomes differentiate into procyclic forms, for development and transmission through the vector. Inhibition of either of these steps by a molecule produced by an altered symbiont could greatly affect establishment, differentiation and transmission of the parasite, and therefore decrease disease prevalence. S. glossinidius can be efficiently cloned and grown in vitro and has already been genetically engineered to produce a trypanocidal cationic peptide attacin, which inhibits growth of both BSF and procyclic forms in vitro and in vivo (Hu, 2005). This has so far proven to be a very promising therapeutic approach for the future control of trypanosomiasis (Hu, 2005).
111. Trypanosome biology
African trypanosomes are amongst the earliest eukaryotic microorganisms. They are .single celled, flagellated protists of the kinetoplastid group of protozoa. These parasites are organisms that complete part of their life cycle in a mammalian host and part in a tsetse fly vector. These organisms have gained notoriety, not only because they
yL--- Animchondrion Endoplasmic retkdurr
Golgi appar-8
k-1-
Receptor-mediated endocyrosit from flagellar pocketGlpmsome
Figure 2. Principal cellular structures of
Trypanosoma
brucei depicted asa
partial longitudinal section. (Vickerman, 1993)cause devastating disease
in
both humans and animals, but because they have the unique capacity to adapt to a huge variety of environments, illustrating the capabilities of a single cell (Maga, 1999).i. Parasite molecular biology
At each life cycle stage AlXcan trypanosomes can be characterized by the cell shape, size and motility, metabolism and surface coat (Gull, 200 1). The parasites have an elongated shape that changes in size fiom a long slender form in the bloodstream of a mammalian host to the short stumpy forms that establish infection in the tsetse fly vector. The cell shape is conferred by a network of microtubules that underlie the entire plasma membrane, with the exception of a small invagination at one end, termed the flagellar
pocket, from which the flagellum emerges (Bangs, 1998; Maga, 1999). All trypanosomes have a nucleus, rough and smooth endoplasmic reticulum, a single morphologically identifiable Golgi apparatus, small as well as large lysosomal-like vesicles, flagellum, mitochondrion and a specialized peroxisome-like organelle termed the glycosome (Figure 2) (Clayton, 1995; Bangs, 1998; Maga, 1999; Cross, 2001; Gull, 2001). African trypanosomes are strictly extracellular pathogens, using their single flagellum for motility. However, they do exhibit some stationary stages in the vector; they form interdigitations with epithelial cell microvilli in the midgut and hemidesmosomal-like attachments on a variety of tissues via their flagellum, more specifically, the paraflagellar rod proteins that extend along the length of the flagellum (Maga, 1999). Kinetoplastids were amongst the earliest organisms to have either a mitochondrion or peroxisome (Clayton, 1995). The nucleus of T. brucei contains a genome comprised of 11 diploid megabase chromosomes, a set of intermediate-sized chromosomes and a large number of mini-chromosomes (McCulloch, 2004). Extensive vesicular trafficking is seen between the flagellar pocket and the nucleus where the single Golgi apparatus is situated (Figure 2). The flagellar pocket is the only site of endocytosis and exocytosis in the bloodstream form of the parasite because it is the only site on the cell surface that is not underlined by the microtubular network (Clayton, 1995; Bangs, 1998; Maga, 1999). Endocytosis and exocytosis occur at much higher rates in the BSF, probably due to the high turnover rate of the major variant surface glycoprotein (VSG). The flagellar pocket also seems to be the only area on the cell surface where membrane bound, surface receptors are exposed to the extracellular environment (Pays et al., 1998). The flagellum is anchored in the cell by the basal body which physically interacts with the mitochondrion. The mitochondrion is
a branched network of cristae that enlarges during differentiation into the procyclic stage (Clayton, 1995). A complex network of mitochondria1 DNA is concentrated in the region closest to the flagellar pocket and is called the kinetoplast (Figure 2) (Clayton, 1995). The kinetoplast consists of a network of thousands of topologically interlocked DNA
circles and is a unique structure (Clayton, 1995). There are about 50 copiedcell of 20-40 kb maxicircle DNA and 5,000-10,000 copiedcell of 0.65-2.5 kb minicircle DNA in the kinetoplast (Clayton, 1995; Gull, 200 1). Mitochondria1 mRNAs undergo a unique form of post-transcriptional modification, termed RNA editing. During this event uridylate residues are inserted and deleted from mRNAs to produce mature mRNA molecules (Clayton, 1995; Cross, 2001; Gull, 2001; Gull, 2003).
African trypanosomes have become very well known for some unique characteristics with respect to their metabolism and surface coat proteins that both change depending on the life cycle stage of the parasite (Bangs, 1998). These include the enzymes required for production of ATP via glycolysis. The seven glycolytic enzymes responsible for converting glucose to 3-phosphoglycerate are housed in a specialized organelle called the glycosome. The other glycolytic enzymes are contained in the cytoplasm where the Krebs cycle and respiratory chain enzymes are also located (Vickerman, 1993; Clayton, 1995; Cross, 2001; Gull, 2003). In the bloodstream, the parasites exploit the glucose rich environment; however, in the tsetse fly midgut proline becomes the main energy source. Throughout the life cycle the parasites express a unique cell surface coat that forms the first line of contact with both the host and vector (Bangs, 1998). In the bloodstream, parasites undergo antigenic variation by replacing their VSG coat to escape the host immune system. The procyclic forms express two
different major forms of procyclins, thought to be involved in protease protection, differentiation and tropism through the vector.
ii. Parasite life cycle in the tsetse fly vector
Trypanosomes exhibit a complex, digenetic life cycle that alternates between the tsetse fly vector and a mammalian host (Figure 3). The life cycle of trypanosomes within the vector begins when a tsetse fly takes a bloodmeal fiom an infected mammalian host and ingests infective BSF trypanosomes. Differentiation and migration, fiom the midgut to the salivary glands, completes this part of the life cycle. This can span a period of a few days in T. vivax to a few weeks in T. brucei sspp. (Aksoy et al., 2003). Once ingested by the fly, the trypanosomes must quickly adapt to a completely new and hostile environment, to do this they undergo morphological and biochemical changes to survive (Treumann et al., 1997). They must transform in order to grow and divide at a lower temperature (27 OC as opposed to 37 OC), utilize a different food source and protect themselves against destructive digestive enzymes and immune molecules (Aksoy et al., 2003). This transformation can be induced in vitro by triggering the citric acid cycle via the addition of cis-aconitate or citrate (Vassella et al., 2003). However, it is thought that these metabolites are not involved in vivo (Vassella et al., 2001; Aksoy et al., 2003). Other triggers of differentiation include temperature drop, trypsin and mild acid stress. Of these, only the temperature drop is known to be experienced by the trypanosomes in vivo, in the fly. The BSF are ingested first into the crop, and then enter the midgut lumen.
IN TSETSE SAUMRY GLANDS
Figure 3. Schematic diagram of the digenetic life cycle
of
Afi-ican bypanosomes. The diagram shows changes in cell surface, mitochondrion, glycosomes, receptor- mediated endocytosis and the relative size of the different stages. The VSG coat is shed concurrently with thegain
of procyclins upon entry into the tsetse midgut. VSG is regained in the metacyclic stage inthe:
salivary gland. Division occurs in the slender fom in the mammal and the procyclic form in the tsetse midgut (Vickerman, 1985).slender BSF usually die and the short-stumpy forms differentiate into procyclic trypanosomes in endoperitrophic space of the midgut (Vickerman 1985; Vickerman; 1993). The trypanosomes undergo critical transformations within the first 24-72 hours, in order to survive and develop in their new host (Vickerman, 1985). The most apparent change is the loss of the VSG surface coat and its successive replacement with procyclins. The transforming trypanosomes increase in body length, while the mitochondrion expands into a branched network with discoid rather than tubular cristae and increases in size by 5% to 25%. Concomitantly, the glycosomes change fiom spherical shapes to bacilliform structures and endocytosis ceases (Figure 3). This is all accompanied by rapid division of the flagellates (Vickerman, 1985). Biochemically, the procyclic forms switch from using glucose to proline as the primary energy source. Activation of the mitochondrion is also associated with a switch to cytochrome-mediated terminal respiration and this may correlate with the change in form of the mitochondria1 cristae. From four days onwards, the trypanosomes actively invade the ectoperitrophic space between the peritrophic matrix and the gut epithelium (Vickerman, 1985; Acosta- Serrano et al., 2001). Within the ectoperitrophic space, rapid division occurs over the next week and this space becomes densely packed with parasites. As the parasites move forward to the proventricular space they grow very long, (up to 60 pm), cease to divide and are termed mesocyclic trypanosomes (Figure 3). It is thought that these parasites are the ones to reinvade the endotrophic space in order to undergo migration to the salivary glands via the oesophagus, mouthparts and salivary ducts (Vickerman, 1985). Infection is established within the salivary glands, where four different life cycle stages develop. The epimastigotes are the main proliferative stage and become attached to the microvillae
via the flagellum. Attachment appears to be essential for the generation of infective metacyclics. Transformation from the epimastigote to the metacyclic stage occurs via two intermediate stages. The attached epimastigotes lose their procyclin surface coat and transform into premetacyclics. These parasites still divide, and retain the branched mitochondria and bacilliform glycosomes, however, the flagellipodia are greatly reduced. The nascent metacyclic forms cease division and detach from the microvillae. Transformation into mature metacyclics is accompanied by, re-acquisition of the VSG surface coat, unbranched mitochondria and spherical glycosomes. This indicates a change in metabolism, in preparation for survival within the mammalian host (Figure 3) (Vickeman, 1 985).
The advantages gained by the trypanosome during its life cycle within the fly include: amplification of a typically sparse parasitaemia in the host, the potential transfer of infective forms every time the fly takes a bloodmeal from a new host, and the opportunity for sexual reproduction (Aksoy et al., 2003). Taking the whole life cycle into consideration, the passage through the fly plays a critical role and is essential in maintaining species integrity, apart from being a convenient mode of transmission (Aksoy et al., 2003).
iii. Parasite life cycle in the mammalian host
The trypanosome life cycle within the mammalian host begins when an infected tsetse fly bites and takes a bloodmeal. During this transaction saliva and non-dividing metacyclic trypanosomes from the salivary gland enter into the skin (Vickerman, 1985; Matthews et al., 2004). Trypanosomes induce an inflammatory reaction at the site of
infection, and a skin chancre develops that resolves within one month (Vickerman, 1993). From the initial site of infection the parasites enter the draining lymph nodes and then the bloodstream. They travel through blood vessels and lymphatic capillaries into connective tissue and eventually enter the brain and cerebrospinal fluid (Vickerman, 1985). Within the mammalian host they are subjected to specific and non-specific immune responses and have therefore evolved complex strategies with which to evade eradication from the host (McCulloch, 2004).
The VSG-expressing metacyclic forms must quickly transform into BSF in order to survive within the host (Vickerman, 1985; Matthews et al., 2004). This includes replacing the metacyclic VSGs with BSF VSGs. BSF trypanosomes exploit the abundance of glucose found in the bloodstream of mammalian hosts and this becomes their primary energy source. The bloodstream population is described as pleomorphic for it contains slender forms, stumpy forms, and transitional forms at different intermediate stages between the latter two (Figure 3) (Matthews et al., 2004). The transition between dividing, slender forms, and the non-dividing, stumpy forms, requires progression from proliferation to cell cycle arrest. This is accompanied by a number of morphological and biochemical transformations. Key features of the stumpy form include: cell cycle arrest, elaboration of some mitochondria1 functions, and relative resistance to antibody-mediated lysis. If a bloodmeal is taken from an infected host it is the stumpy forms that will progress to the next stage in the life cycle within the tsetse fly midgut (Figure 3) (Matthews et al., 2004).
African trypanosomes have developed mechanisms to disrupt and inhibit host defenses. Investigation into the mechanisms used by African trypanosomes to evade the
immune responses of their mammalian hosts began in the early nineteen hundreds. Infected mammals produce antibodies to a highly immunogenic surface molecule (Black
et al., 200 1 ; Morgan et al., 2002). In 1907 an Italian scientist, A. Massaglia, concluded that African trypanosomes in the bloodstream escaped destruction because they adapted to interfere or block the action of antibodies (Donelson, 1998). In 1975, Cross described heterogeneous surface glycoproteins, expressed by BSF trypanosomes, which induced clone-specific immunity in infected mice (Cross, 1975). This article proved that seventy years earlier Massaglia had come to the right conclusion. This unique technique of switching surface coats to evade host immune responses was termed antigenic variation. BSF trypanosomes have become famous, because much research has gone into elucidating the molecular mechanisms responsible for this phenomenon (Donelson, 1998). Individual trypanosomes express one VSG at a time, therefore the mammalian host experiences recurring waves of parasitaemia because the main immune response is against the VSG which changes with each new wave. A VSG specific response takes many days to develop, therefore the host experiences high levels of parasitemia due to the quick doubling time of trypanosomes (approximately 7 hours). This leads to severe pathology, immunosuppression and eventually debilitating secondary infections in the host (Black et al., 2001).
Antigenic variation is not the sole factor responsible for immune evasion and maintenance of a chronic infection (Black et al., 2001; Morgan et al., 2002). As BSFs are lysed and destroyed throughout an infection the immune system is assaulted not only by different classes of VSGs but also by invariant, intracellular antigens. Parasite levels increase dramatically upon initial infection inducing a massive, non-specific expansion of
polyclonal B-cells. These B-cells produce immunoglobulin isotype M (IgM), antibodies, that recognize parasite antigens, which may also cross-react with self-antigens (Donelson, 1998; Pays et al., 1998). The huge IgM response is not followed by the typical concomitant increase in immunoglobulin isotype G (IgG) and other antibody classes. Suppression of many B-cell and T-cell responses is accompanied by the suppression of other secondary immune events. Fore example, interferon gamma production is increased, and may correlate with increased macrophage activity. Interleukin-2 levels are decreased which in turn may explain the lack of T-cell proliferation. Parasites appear to evade complement-mediated lysis by opsonization and destruction by liver macrophages (Donelson, 1998).
IV. The importance of trypanosome cell surface molecules
The trypanosome surface is the first interface to come in contact with the hostile environment of the mammalian bloodstream or the insect midgut. Therefore, the major surface molecules coating the bloodstream form (mammalian) and procyclic form (insect) trypanosomes are the first line of defense against the host immune system. The major surface glycoproteins of BSF and procyclic form, likely play a role in establishment of infection, parasite development and differentiation. In addition to the major surface glycoproteins, less accessible, underlying surface molecules are also present, and may provide essential functions for parasite viability. The critical role surface molecules play in the life cycle and parasitaemia of African trypanosomes makes these molecules attractive research subjects. Chapter 2 will discuss the major and minor surface molecules expressed by Afiican trypanosomes that are known today. What follows is a
molecular and biochemical study of the surface proteins expressed by wild type PCF
T.
b. brucei and three knock out PCF mutants that differ in their surface protein expression.Chapter 2. The surface coat of PCF African trypanosomes
1. Introduction
The surface coat of African trypanosomes is comprised of two major surface proteins. As trypanosomes move between hosts in their life cycle and go through their different developmental stages the expression of these two surface molecules changes as previously discussed in Chapter 1. Chapter 2 begins with a closer look at the structure and regulation of these surface molecules.
1.1 Variant surface glycoproteins of bloodstream forms of African trypanosomes
i. Regulation of VSG gene transcription
Bloodstream forms of African trypanosomes are infamous for their ability to show antigenic variation which allows them to avoid immune elimination. In a population of parasites, there is sequential expression of individual members of a large repertoire of variant surface glycoprotein (VSG) genes that encode antigenically distinct proteins (Donelson, 1998).
The trypanosome genome contains several hundred genes encoding immunologically distinct VSGs. To ensure that an individual cell expresses only one VSG, there are multiple sites of transcription, and only one is actively transcribed at a time (Ferguson, 1997). Large numbers of silent VSG genes are also spread throughout the genome. There are tens of VSG expression sites, and hundreds of silent VSG genes (McCulloch, 2004). There is strong evidence to indicate that the general pathways of
homologous recombination and repair are responsible for VSG switching. It is thought that a gene copy is generated from a silent VSG gene and then moved into an active expression site (ES), deleting the resident VSG gene and leaving the formerly silent one to be expressed (Pays et al., 1998; McCulloch, 2004). Within the mammalian bloodstream,
T.
brucei expresses a class of VSG expression sites called BSF ESs, which have complex structures containing Expression Site-Associated Genes (ESAGs) that are co-transcribed along with the VSG gene. In the salivary gland, where trypanosomes differentiate into infective metacyclics, a class of VSG genes called telomeric metacyclic ESs are active so that the parasite can reacquire its VSG coat (McCulloch, 2004).ii. Structure of VSG proteins
Bloodstream forms express an unusual surface coat composed of about one million copies of VSG glycoproteins arranged as a dense monolayer of homodimers on the parasite surface (Figure 4) (Ferguson, 1997; Mehlert et al., 1998; Pays et al., 1998). Each VSG molecule has an approximate mass of 55 kDa. The amino terminal domain of 350 to 400 residues represents about 75% of the polypeptide and is highly variable (Mehlert et al., 1998; Pays et al., 1998). However, there is some sequence similarity amongst VSG N-terminal domains implying that some structure is conserved. X-ray crystallography studies showed that different VSG polypeptides fold into similar elongated shapes, with two extended anti-parallel alpha-helical bundles per monomer fonn'ing a 15 nm thick coat (Figure 4) (Clarke, 1988; Ferguson, 1997; Mehlert et al., 1998; Pays et al., 1998). In 1988 Clarke and colleagues showed that the antigenic
Figure 4. Simple model of the major surface molecules of bloodstream form and procyclic form T. brucei. VSGs form a dense surface coat on BSF in comparison to the more diffuse surface coat of PCF comprised of procyclins. VSG dimers sit on top of a glycocalyx made up of the glycans and GPI anchors. Procyclins form rod like structures attached to the membrane via GPI anchors that are highly glycosylated. (Mehlert et
al., 1998)
differences between VSGs were dependent on topographical assembled epitopes on the parasite surface of epitopes that were highly sensitive to structural changes (Clarke, 1988). All VSGs are post-translationally N-glycosylated, typically once or twice, at or near the C-terminal domain (Ferguson, 1997; Pays et al., 1998). All VSGs are attached to the plasma membrane through a covalent linkage between the C-terminal amino acid and a glycosylphosphatidylinositol (GPI) membrane anchor (Pays et al., 1998). Mature VSG GPI anchors contain a common core structure of ethanolamine-HP04-6Mana1- 2Mancll-6Manal-4GlcNa 1 -6PI characteristic to most GPI anchors. However, this anchor is then modified by a galactose side chain that is unique to VSGs. The 3-D structure predicts that the anchor forms a large, dense, plate-like calyx of carbohydrate on which the VSG polypeptide sits (Ferguson, 1997).
iii. Functions of VSG
The VSG proteins are so densely packed that a very limited stretch of N-terminal amino acid sequence is accessible to the extracellular environment and all of the C- terminus is buried (Mehlert et al., 1998; Pays et al., 1998). The dense packing combined with the overall thickness of the VSG coat means that only a limited subset of B-cell- stimulating epitopes are exposed on the surface (Ferguson, 1997; Pays et al., 1998; McCulloch, 2004). VSGs are highly immunodominant and the mammalian host recognizes and mounts an immune response to these proteins. Therefore the VSG coat shields underlying invariant surface proteins that would be potential targets for attack by the immune system (Pays et al., 1998). Consequently, antigenic variation of VSGs and their densely packed surface expression enables some parasites to evade both specific humoral immune responses as well as components of the alternative complement pathway (Mehlert et al., 1998). VSG switching occurs at a frequency of lom2 per cell and per generation so that the parasites can maintain a persistent infection rather than be completely eliminated (Pays et al., 1998; McCulloch, 2004).
1.2 Procyclins expressed by PCF African trypanosomes
Although many laboratories have been engaged in research to develop a vaccine against BSFs of African trypanosomes, this has proved frustrating, and to date, htile. Consequently, some research groups have become more interested in studying the procyclic form of the parasite and tackling the biology of parasite-vector interactions. The interaction between parasite and host molecules within the tsetse fly vector are vital, and offer potential control strategies that could limit or halt transmission of the disease.
